The goal of the proposed work is to systematically explore whether and how proteins that sense and shape the curvature of plasma membranes are responsible for building the intricate dendritic and axonal arbors that distinguish neurons from other cell types. The formation of complex 3-dimensional branched membrane structures is one of the most fundamental properties of neurons that enable them to transmit information between neurons and from neurons to other cell types. The ability of selected proteins to sense membrane curvature during this differentiation process is important as defects in proteins, such as Oligophrenin and srGAP2, that can bind to and shape lipid membranes cause neurodegenerative diseases. Our proposal aims to develop and execute a scalable experimental strategy to understand the process of arbor formation by focusing on a family of Bar domain containing proteins that are known from in vitro studies to be able to bind to and shape curved membranes. We will systematically investigate their function in generating the branched extended plasma membrane architecture of neurons. Currently available in vitro assays and structural studies of proteins with membrane binding domains can determine the radius of the membrane curvature that results from the formation of oligomers by curvature sensing proteins. Using this approach, proteins have been identified that sense and shape membranes with positive and negative curvatures. Nevertheless, it is difficult from these assays to know to which curved intracellular membranes these proteins may bind, and if or how they act dynamically to generate distinct types of curved plasma membranes in a living cell. We developed a novel assay to investigate curvature dependent processes that is based on fabricated nanostructures that trigger plasma membrane curvature in living cells. Specifically, our project will deliver a new scalable assay based on these nanostructures that allows one to measure in living cells the intracellular membrane localization and the curvature preference as well as the dynamic assembly, disassembly and exchange rate of curvature sensing membrane binding proteins. Our initial studies already identified and characterized a key regulator that binds to positively curved plasma membranes and is critically involved in controlling neuronal architecture. We have combined this approach with parallel high-throughput live-cell imaging and automated image analysis of cultured hippocampal neurons that enables us to systematically analyze the cellular roles of these same Bar domain binding proteins in controlling the neuronal architecture. At the center of our work is the development of this synergistic dual experimental approach that can ultimately be used as an unbiased and systematic platform to investigate the neuronal roles of a large number of putative neuronal membrane binding proteins. Together, our project will provide a molecular framework to understand the program used by neurons to create the vast repertoire of different neuronal architectures. PUBLIC HEALTH RELEVANCE: Project Narrative The long term goal of the project is to understand the cellular mechanism of how neurons build the intricate dendritic and axonal branch structure that distinguishes them from other cell types. We will develop and execute a scalable experimental strategy to understand how large families of proteins that sense and change the curvature of the plasma membrane synergistically control the neuronal architecture.